Water status and thermal analysis of alginate beads used in cryopreservation of plant germplasm.

Encapsulation and dehydration techniques using alginate beads are used increasingly for the pre-treatment of various plant materials for cryopreservation to improve survival post-cryogenic storage. This study reports the effects of the water content of beads (formed with 3% (w/v) alginic acid in liquid S-RIB), polymerisation time (in 100 mM calcium chloride solution), osmotic dehydration (in 0.75 M sucrose solution), and evaporative air desiccation on the thermal properties of alginate beads used in cryopreservation protocols. Experimental beads were assayed using a differential scanning calorimeter (DSC) with a cooling programme to -150 degrees C, followed by re-warming. Resultant thermograms were evaluated with particular reference to the onset temperature and enthalpy of the melt endotherm from which the quantities of frozen and unfrozen water were calculated. Treatments were applied sequentially to samples of beads and their thermal features evaluated at each stage of the protocol. Using 'standard' beads (40-55 mg fresh weight), formed using plastic disposable pipettes, the degree of polymerisation (>10 min) proportionally reduced their dry weight and increased their water content. Thermal characteristics of the beads were unaffected by polymerisation times >10 min, but the maximum level (23%) of unfrozen (osmotically inactive) water was achieved after 15 min polymerisation. Osmotic dehydration using 0.75 M sucrose significantly lowered bead water content and mean dry weight approximately doubled with 20-24 h immersion time. Bead desiccation in still air reduced their water content by 83% of fresh weight, whilst dry weight remained constant. After 8 h desiccation in air between 27 and 37% of the water in the bead was osmotically inactive (unfrozen) in DSC scans. Desiccation >18 h reduced this fraction to zero. The melt onset temperature and the enthalpy of melting were directly related to bead water content. The unfrozen water fraction increased substantially with reduced water content of the beads (from 23 to 37% of total water content), concomitant with a reduction in the ratio of unfrozen to frozen water from 1:3 to 1:2. For successful vitrification and the production of a glass that did not destabilise on rewarming, a bead water content of ca. 26% of fresh weight (0.4 g waterg(-1) dry weight) was required, much of which was osmotically inactive water. These data are discussed in relation to optimal pre-treatments for alginate bead encapsulation techniques used in the cryopreservation of a range of plant germplasm. It is proposed that increased standardisation of alginate beads, in terms of volume, fresh weight, and water content, is required to reduce the variability in physical and thermal features, which in turn will improve survival of plant samples post-cryopreservation.